A Cooperative Folding Unit as the Structural Link for Energetic Coupling within a Protein

Abstract

Previously, we demonstrated that binding of a ligand to Escherichia coli cofactor-dependent phosphoglycerate mutase (dPGM), a homodimeric protein, is energetically coupled with dimerization. The equilibrium unfolding of dPGM occurs with a stable, monomeric intermediate. Binding of several nonsubstrate metabolites stabilizes the dimeric native form over the monomeric intermediate, reducing the population of the intermediate. Both the active site and the dimer interface appear to be unfolded in the intermediate. We hypothesized that a loop containing residues 118-152 was responsible for the energetic coupling between the dimer interface and the distal active site and was unfolded in the intermediate. Here, we investigated the structure of the dPGM intermediate by probing side-chain interactions and solvent accessibility of the peptide backbone. By comparing the effect of a mutation on the global stability and the stability of the intermediate, we determine an equilibrium φ value (φ value), which provides information about whether side-chain interactions are retained or lost in the intermediate. Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) was used to investigate differences in the solvent accessibility of the peptide backbone in the intermediate and native forms of dPGM. The results of φ value analysis and HDX-MS reveal the least stable folding unit of dPGM, which is unfolded in the intermediate and links the active site to the dimer interface. The structure of the intermediate reveals how the cooperative network of residues in dPGM gives rise to the observed energetic coupling between dimerization and ligand binding.

Identification of Novel Protein Lysine Acetyltransferases in Escherichia coli

“…While this enzyme was not previously considered to be allosteric, it was recently proposed to function as an allosteric enzyme, whereby dimer stabilization acts as the allosteric signal that is transmitted rather than the more typical binding of a specific effector to an allosteric site. In this case, the ordering and stabilization of the region that contains the lysines acetylated by AcP act as the transmission signal ( 34 , 35 ). While all AcP acetylations occurred on this highly flexible domain of the protein, the KAT acetylation site (K86) was found outside the active site on a small 3 10 -helix near but not directly interacting with the opposite monomer at the dimer interface.…”

Towards understanding the novel adhesin function of Candida albicans phosphoglycerate mutase at the pathogen cell surface: some structural analysis of the interactions with human host extracellular matrix proteins

“…The structure of Gpm1 is well known, but molecular analyses, aimed at locating binding sites for various ligands have not been performed so far. One study showed a possible important function of ATP in stabilizing the structure and dimerization of the Gpm1 molecule (Chang et al, 2012;Gardner et al, 2017). Our study on the interactions between Gpm1 and ECM proteins revealed possible interaction sites at motifs aa 116-158 for VTR and aa 138-175 for FN, with aa 138-158 sequence binding to both host proteins.…”

Identification of novel protein lysine acetyltransferases inEscherichia coli

“…While this enzyme was not previously considered to be allosteric, it was recently proposed to function as an allosteric enzyme, whereby dimer stabilization acts as the allosteric signal that is transmitted rather than the more typical binding of a specific effector to an allosteric site. In this case, the ordering and stabilization of the region that contains the lysines acetylated by AcP acts as the transmission signal (34, 35). While all AcP acetylations occurred on this highly flexible domain of the protein, the KAT acetylation site (K86) was found outside the active site on a small 3 10 -helix near but not directly interacting with the opposite monomer at the dimer interface.…”